Journal: Cell Communication and Signaling : CCS

Article Title: Acquired radioresistance in cancer associated fibroblasts is concomitant with enhanced antioxidant potential and DNA repair capacity

doi: 10.1186/s12964-021-00711-4

Figure Lengend Snippet: Oxidative stress in AG1522 CAFs that were cocultured with MDA-MB-231 or MCF7 breast cancer cell for 120 h. a Protein carbonylation demonstrating alterations in protein oxidation with both increased (~ 40 kDa) and decreased (~ 60 kDa) bands. b Induction of mitochondrial oxidative stress: the prevalence of superoxide anion (O 2 ·− ) was analyzed by MitoSOX™ Red and flow cytometry in AG1522 CAFs cocultured with MDA-MB-231 (light green histogram) or MCF7 (dark green histogram) breast cancer cells, and in AG1522 fibroblasts cocultured with MCF10A (purple histogram) non-tumorigenic breast epithelial cells or themselves (orange histogram) (control). Additional controls consisted of AG1522 fibroblasts without MitoSOX™ Red loading (red histogram) (negative control) and AG1522 fibroblasts treated with antimycin A (blue histogram) (positive control). AG1522 CAFs cocultured with MDA-MB-231 (light green) show a ten-fold increase in MitoSox™ levels over negative control (red). The histograms corresponding to AG1522 cocultures with MCF7 (dark green), MCF10A (purple), or AG1522 (orange) possess subpopulations of cells with a higher expression of MitoSOX™ Red (see appearance of bumps in tails of descending parts of the curves compared to the smoother lognormal shape in the negative control) than the larger population; however, no other significant changes were observed for these samples. c , d Modulation of antioxidant enzyme activity. Antioxidant enzyme activities in c AG1522 CAFs and d MDA-MB-231 cells cocultured with each other for 5, 48 or 120 h. Coculture with MDA-MB-231 resulted in an increased in MnSOD activity in AG1522 CAFs at 48 and 120 h. Additionally, although the enzyme activity remained unchanged, the catalase band appeared to run slower through the gel in AG1522 CAFs at 48 and 120 h. No changes were noted in CAF CuZnSOD or any of the antioxidant enzymes in the MDA-MB-231 lysate samples. Fold change = relative change. The results are representative of two separate experiments. In each experiment, 5 independent replicates were combined to generate enough cells for analysis

Article Snippet: Human breast non-tumorigenic epithelial MCF 10A cells (MCF10A) (ATCC, CRL-10318) were grown in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12) (Corning CellGro) supplemented with 5% (vol/vol) horse serum (Invitrogen), 2 mM l -alanyl- l -glutamine, 2.5 μg/mL Amphotericin b, 0.02 μg/mL EGF, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 0.1 μg/mL cholera toxin (Sigma), and 100 units/mL penicillin and 100 μg/mL streptomycin.

Techniques: Flow Cytometry, Control, Negative Control, Positive Control, Expressing, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Acquired radioresistance in cancer associated fibroblasts is concomitant with enhanced antioxidant potential and DNA repair capacity

doi: 10.1186/s12964-021-00711-4

Figure Lengend Snippet: Increased radioresistance of AG1522 CAFs requires coculture with cancer cells versus epithelial cells. Micronucleus formation in AG1522 cells cocultured for 120 h with MCF7 breast cancer cells or MCF10A non-tumorigenic breast epithelial cells, followed by exposure to 0.5 Gy of 137 Cs γ-rays. Fold changes in micronuclei are normalized to respective sham-irradiated (0 Gy) control. Coculture with MCF10A failed to protect the AG1522 fibroblasts from the radiation insult, whereas coculture with MCF7 significantly reduced the levels of micronuclei in irradiated CAFs. The results are representative of three separate experiments

Article Snippet: Human breast non-tumorigenic epithelial MCF 10A cells (MCF10A) (ATCC, CRL-10318) were grown in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12) (Corning CellGro) supplemented with 5% (vol/vol) horse serum (Invitrogen), 2 mM l -alanyl- l -glutamine, 2.5 μg/mL Amphotericin b, 0.02 μg/mL EGF, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 0.1 μg/mL cholera toxin (Sigma), and 100 units/mL penicillin and 100 μg/mL streptomycin.

Techniques: Irradiation, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Acquired radioresistance in cancer associated fibroblasts is concomitant with enhanced antioxidant potential and DNA repair capacity

doi: 10.1186/s12964-021-00711-4

Figure Lengend Snippet: Increased radioresistance of MRC5 CAFs is cancer cell dependent. Micronucleus formation in MRC5 CAFs cocultured for 120 h with MDA-MB-231 or MCF7 breast cancer cells, or MRC5 fibroblasts cocultured with MCF10A non-tumorigenic breast epithelial cells, followed by exposure to 1 Gy of 137 Cs γ-rays. The cells were γ-irradiated while in coculture. Fold changes in micronuclei are normalized to respective sham-irradiated (0 Gy) control. Only MRC5 CAFs cocultured with MDA-MB-231 demonstrated significantly increased resistance to ionizing radiation, when compared to control ( p < 0.05). The results are representative of three separate experiments

Article Snippet: Human breast non-tumorigenic epithelial MCF 10A cells (MCF10A) (ATCC, CRL-10318) were grown in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12) (Corning CellGro) supplemented with 5% (vol/vol) horse serum (Invitrogen), 2 mM l -alanyl- l -glutamine, 2.5 μg/mL Amphotericin b, 0.02 μg/mL EGF, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 0.1 μg/mL cholera toxin (Sigma), and 100 units/mL penicillin and 100 μg/mL streptomycin.

Techniques: Irradiation, Control

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet: ( A ) Western blot analysis of indicated proteins in Lig4 − /− , Lig4 − /− : Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells. ( B ) Flow cytometric analysis of chromatin-bound RPA (top) and γH2AX (bottom) before and after IR of non-cycling Lig4 − /− , Lig4 − /− : Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells. The experiments were repeated in two independently generated cell lines at least twice. ( C ) Western blot analysis of RPA32 and phosphorylated RPA 32 (pRPA32(S4/S8)) of non-cycling Lig4 − /− , Lig4 −/− :Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells treated without or with IR after indicated times. ( D ) Flow cytometric analysis of cycling and non-cycling Lig4 − /− , Lig4 − /− : Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells for BrdU incorporation and DNA content (7-AAD). Percentage of cells in S-phase is indicated. ( E ) Representative images of IR-induced RPA foci in non-cycling Lig4 − /− , Lig4 − /− : Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells from two independent experiments. ( F ) Western blot analysis of indicated proteins in WT, Trp53bp1 − /− , and Lin37 − /− MCF10A cells. ( G ) Flow cytometric analysis of BrdU pulsed cycling (top) or non-cycling (bottom) WT, Trp53bp1 − /− , and Lin37 − /− MCF10A cells as in ( D ). ( H, I ) Flow cytometric analysis of chromatin-bound RPA (top) and γH2AX (bottom) before or after IR of non-cycling WT and ( H ) Trp53bp1 − /− or ( I ) Lin37 − /− MCF10A cells. IR, ionizing radiation; WT, wild type.

Article Snippet: WT and Lig4 −/− abl pre-B cells and MCF10A human mammary epithelial cells (from ATCC, CR-10317) used in this study all contain pCW-Cas9 (Addgene# 50661), which has a FLAG-tagged Cas9 cDNA under the control of a doxycycline-inducible promoter.

Techniques: Western Blot, Generated, BrdU Incorporation Assay

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet: ( A ) Western blot analysis (top) and flow cytometric analysis for chromatin-bound RPA after before or after IR (bottom) of non-cycling Lig4 − /− : Lin37 − /− abl pre-B cells with empty lentivirus or lentivirus expressing wild type (WT) LIN37 or the LIN37 (CD) mutant. Representative of three experiments. ( B ) Volcano plot of RNA-Seq analysis of non-cycling Lig4 − /− and Lig4 − /− : Lin37 − /− abl pre-B cells showing log2 values of the ratio of normalized transcript levels of Lig4 −/− :Lin37 −/− to Lig4 −/− cells (X-axis) and −log10 of the q-values of fold enrichment of each gene (Y-axis). The dashed line indicates q=0.05. Genes with q-values≤0.05 are denoted as red dots. ( C ) Western blot analysis of indicated proteins in cycling and non-cycling Lig4 − /− , Lig4 − /− : Trp53bp1 − /− , and Lig4 − /− : Lin37 − /− abl pre-B cells. ( D ) Western blot analysis of indicated proteins in cycling or non-cycling WT , Trp53bp1 − /− , and Lin37 − /− MCF10A cells. ( E ) Western blot analysis of indicated proteins in cycling and non-cycling Lig4 − /− : Lin37 − /− abl pre-B cells with empty lentivirus or lentivirus expressing WT LIN37 or the LIN37 (CD) mutant. HR, homologous recombination; IR, ionizing radiation; RNA-Seq, RNA sequencing. Figure 5—source data 1. RNA-Seq result and GO analysis in non-cycling Lig4 − /− and Lig4 − /− : Lin37 −/− abl pre-B cells. GO, gene ontology; RNA-Seq, RNA sequencing.

Article Snippet: WT and Lig4 −/− abl pre-B cells and MCF10A human mammary epithelial cells (from ATCC, CR-10317) used in this study all contain pCW-Cas9 (Addgene# 50661), which has a FLAG-tagged Cas9 cDNA under the control of a doxycycline-inducible promoter.

Techniques: Western Blot, Expressing, Mutagenesis, RNA Sequencing, Homologous Recombination

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet: ( A, B ) Western blot analysis of indicated proteins in cycling and non-cycling abl pre-B cells ( A ) or MCF10A cells ( B ). ( C, D ) Western blot analysis of indicated proteins in cycling G 1 or S/G 2 /M abl pre-B cells ( C ) or MCF10A cells ( D ), isolated by flow cytometric cell sorting based on the PIP-FUCCI reporter. Representative of two independent experiments. Asterisk indicates non-specific recognizing bands. ( E, F ) Flow cytometric analysis of chromatin-bound RPA and γH2AX before and after IR treatment of G 1 -phase Lig4 −/− , Lig4 −/− :Trp53bp1 − /− and Lig4 −/− :Lin37 − /− abl pre-B cells ( E ) or WT, Trp53bp1 − /− , and Lin37 − /− MCF10A cells ( F ). Representative of three experiments. IR, ionizing radiation; WT, wild type  . Figure 7—source data 1. RNA-Seq result and GO analysis in cycling G 1 Lig4 − /− and Lig4 − /− : Lin37 −/− abl pre-B cells. GO, gene ontology; RNA-Seq, RNA sequencing. Figure 7—source data 2. GO analysis of genes upregulated in non-cycling G 0 and/or cycling G 1 Lig4 − /− : Lin37 −/− abl pre-B cells. GO, gene ontology.

Article Snippet: WT and Lig4 −/− abl pre-B cells and MCF10A human mammary epithelial cells (from ATCC, CR-10317) used in this study all contain pCW-Cas9 (Addgene# 50661), which has a FLAG-tagged Cas9 cDNA under the control of a doxycycline-inducible promoter.

Techniques: Western Blot, Isolation, FACS, RNA Sequencing

Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet: ( A, B ) Flow cytometric analysis of EdU incorporation and DNA content (7-AAD) of cycling Lig4 −/− , Lig4 −/− :Trp53bp1 − /− , and Lig4 −/− :Lin37 − /− abl pre-B cells ( A ) or cycling WT, Trp53bp1 − /− , and Lin37 − /− MCF10A cells ( B ). Cells were treated (+EdU) or not treated (−EdU) with EdU and analyses were carried out before or after IR. The percentage of G 1 -phase cells is shown. IR, ionizing radiation; WT, wild type.

Article Snippet: WT and Lig4 −/− abl pre-B cells and MCF10A human mammary epithelial cells (from ATCC, CR-10317) used in this study all contain pCW-Cas9 (Addgene# 50661), which has a FLAG-tagged Cas9 cDNA under the control of a doxycycline-inducible promoter.

Techniques: